These studies will identify and characterize key components of the intrinsic genetic program that controls development and function of male germ cells and that ultimately define the conditions responsible for male fertility. The approaches currently being applied are to identify genes expressed specifically in male germ cells, use the gene knockout approach to define the roles of the proteins they encode, employ yeast two-hybrid assays and deletion mutagenesis to identify protein-protein interactions essential for development of the male gamete, and prepare antisera to determine the temporal-spatial distribution of specific gene products. Many genes are expressed only in male germ cells and selected genes are being studied that encode proteins whose functions are essential for novel aspects of gamete development and/or function. (1) Initiation and progression of meiosis in male germ cells: Retinoic acid regulates entry into meiosis, but the timing of entry differs between male and female germ cells. We have identified a novel homeobox transcription factor RHOX13 and hypothesize that retinoic acid signaling involves activation of its gene. The Rhox13 gene is transcribed in male and female germ cells beginning on embryonic day E13.5. Translation of RHOX13 occurs in the E13.5 ovary and coincides with the entry of female germ cells into meiosis. Translation of RHOX13 begins on postnatal day P4 and proceeds male germ cells entry into meiosis. We hypothesize that the RNA-binding protein NANOS2 binds to the 3' UTR of the Rhox13 transcript to suppress its translation. Current studies are testing this hypothesis. (2) Regulation of meiotic progression in spermatogenesis: The mechanisms regulating transition from prophase I to metaphase I of meiosis during spermatogenesis are uncertain. Previous studies have suggested that proteins other than or in addition to CDK1 and Cyclin B1 may regulate this process during spermatogenesis. Conditional gene targeting approaches are being used to determine if these proteins are essential for the progression of meiosis in male germ cells. (3) The role of novel scaffold proteins and glycolytic enzymes in sperm motility: The flagellum is one of the most complex of all cell organelles and many of its proteins are encoded by genes expressed during post-meiotic haploid phase of spermatogenesis. We have used a variety of approaches to identify components of the fibrous sheath, a key cytoskeletal component of the sperm flagellum. These included structural proteins, signal transduction anchoring proteins, and glycolytic enzymes, most of which are encoded by genes only expressed in male germ cells. This led us to hypothesize thatmammalian sperm require ATP produced by glycolysis for motility and the fibrous sheath is an essential scaffold for the glycolytic enzymes. This hypothesis has been confirmed using gene targeting and current studies are determining how the different glycolytic enzymes and structural components of the fibrous sheath are assembled and how their functions are regulated.